Biologic tissues depend on the precise regulation of vascular tone and blood flow to maintain the appropriate delivery of oxygen, nutrients, and hormonal signals. Central to the regulation of vascular tone, capacitance and vascular cell biology are the generation of lipid 2nd messengers of signal transduction such as eicosanoids and lysolipids that collectively orchestrate vascular responses. In most cell types, including endothelial cells and smooth muscle cells, the rate-determining step in the production of lipid 2nd messengers is the activity of intracellular phospholipases. During the current grant interval, we have demonstrated the important role of iPLA2-3 in vascular contraction and relaxation using protein, chemical, pharmacologic and genetic approaches. In the vasculature, iPLA2p is inhibited by calcium-activated calmodulin and we have demonstrated that this inhibition can be reversed by acyl-CoA. Moreover, we have demonstrated that iPLA2p is also an acyl-CoA hydrolase and that iPLA2p forms a stable acyl-enzyme at a second active site distinct from the S465 catalytic site. Collectively, these results integrate phospholipase activation and lipid 2nd messenger generation with cellular lipid metabolism to provide an interactive metabolic network regulating vascular tone and vascular biology. These pathways are modulated by acylation, calcium flux and phosphorylation and converge at the level of the iPLA2p protein itself. Accordingly, in Specific Aim 1, we will first determine the molecular mechanisms regulating calcium-independent phospholipase A2 activity by acyl-CoA. Next, we will determine the chemical and physiologic sequelae of covalent acylation of iPLA2p and explore covalent alterations in iPLA2 that affect the kinetic properties and physiologic function of the enzyme in vascular tissues. Multiple complementary approaches will be pursued including tryptic footprinting and cross-linking of signaling complexes with bifunctional cross-linking reagents with analysis by mass spectrometry. In Specific Aim 2, we will identify the intracellular phospholipases A2 responsible for eicosanoid and lysolipid production in the vascular system using genetically altered mice and mass spectrometric identification of alterations in eicosanoid and lipid messengers produced after agonist stimulation. Shotgun lipidomics will facilitate identification of the role of each phospholipase in generating signaling metabolites and modulating specific lipid metabolic fluxes in vascular cells. In Specific Aim 3, physiologic experiments employing in vitro study of mesenteric resistance vessels will determine the mechanisms through which each phospholipase regulates agonist-induced vasoconstriction and vasodilation. Through this multidisciplinary approach, the roles of intracellular phospholipases in vascular function can be determined from detailed chemical mechanisms to in vivo physiology clarifying the functional significance of each intracellular phospholipase in vascular regulation.